Microbiome-mediated Biotransformation of Human Lactoferrin (effera ^® ) Enhances Epithelial Barrier Function in an Ex Vivo Adult Model

Lactoferrin is a multifunctional iron-binding glycoprotein that supports intestinal barrier function, immune regulation, and a favorable gut microbial environment. However, the contribution of gut microbial biotransformation to its gastrointestinal activity remains poorly understood. We investigated whether effera ^® , a precision fermentation-derived recombinant human lactoferrin, supports intestinal barrier function through microbiome-mediated mechanisms. effera ^® underwent simulated upper gastrointestinal digestion followed by ex vivo colonic fermentation using the validated SIFR® technology pipeline, which employs bioreactors that are inoculated with fecal microbiota from six healthy adult donors. Microbial activity was evaluated by measuring short-chain fatty acid (SCFA) production, bacterial cell density, and microbiome composition. effera ^® produced dose-dependent increases in the production of SCFAs and bacterial cell density demonstrating enhanced microbial metabolic activity. These metabolic changes were accompanied by shifts in key microbial groups within Bacillota_A and Bacteroidota. Intact effera ^® and cell-free post-colonic fermentation-derived products were evaluated in a Caco-2/THP-1 epithelial-immune co-culture model under basal and lipopolysaccharide-challenged conditions. Whereas intact protein did not significantly improve epithelial barrier integrity, effera ^® ’s post-colonic fermentation-derived products significantly enhanced transepithelial electrical resistance (TEER) under basal conditions and produced an even stronger barrier-protective response following LPS challenge. Across matched doses, effera ^® consistently generated greater TEER responses than bovine lactoferrin. Improved barrier function was accompanied by increased expression of tight-junction-associated targets ZO-1 and occludin and reduced secretion of CXCL-10 and IL-8. Together, these findings demonstrate that microbial biotransformation enhances the biological activity of effera ^® , linking increased microbial metabolism with improved epithelial barrier integrity and modulation of inflammatory signaling. This integrated study provides a strong mechanistic foundation for the use of human lactoferrin in adult gut-health applications and offers valuable guidance for future adult clinical studies and infant-relevant investigations.